The phenotype of individuals harboring loss-of-function or gain-of-function mutations of the lutropin receptor (LHR) clearly shows that this receptor is important for the proliferation and differentiation of Leydig cells and may even be involved in the neoplastic transformation of this cell type. The experiments proposed herein are driven by the hypothesis that the binding of agonist (LH/CG) to the LHR results in the activation of multiple signaling pathways, and that these pathways, either alone or in combination, stimulate the proliferation of Leydig cells. We seek to identify and characterize novel G protein-dependent and -independent pathways that are activated by the LHR and may control cell proliferation. In pursuing these experiments we will compare the behavior of the hLHR-wt and that of naturally occurring activating mutations of the hLHR associated with Leydig cell hyperplasia or Leydig cell tumors. Some experiments will be done in heterologous cell lines expressing these receptors but we will ultimately make use of the knowledge gained from these models to examine the ability of the LHR to induce the proliferation and neoplastic transformation of progenitor rat Leydig cells in primary culture. The specific aims are as follows. (1) Examine the involvement of G proteins and the beta-arrestins (barrs) on the LH/CG-induced activation of Src, phosphatidylinositol 3 kinase (PI3K) and the mitogen-activated protein kinase (MAPK) cascade. (2) Define the residues of the LHR that bind the barrs and the relative importance of LHR activation vs. phosphorylation on the formation of the LHR/barr complex. (3) Define the molecular basis of the sorting of the internalized LHR and the impact of this sorting on signaling. (4) Compare the pathways involved in the trafficking of the hLHR-wt with the agonist-independent trafficking of activating mutations of the hLHR associated with Leydig cell hyperplasia and Leydig cell tumors. (5) Establish and characterize a new experimental system to test the mitogenic and oncogenic potential of the hLHR.